1. Field of the Invention
The present invention relates to a Corynebacterium glutamicum pyruvate carboxylase protein and to polynucleotides encoding this protein.
2. Background Information
Pyruvate carboxylate is an important anaplerotic enzyme replenishing oxaloacetate consumed for biosynthesis during growth, or lysine and glutamic acid production in industrial fermentations.
The two-step reaction mechanism catalyzed by pyruvate carboxylase is shown below: xe2x80x83ENZ-biotin-CO2+Pyruvatexe2x86x92ENZ-biotin+oxaloacetatexe2x80x83xe2x80x83(2)
In reaction (1) the ATP-dependent biotin carboxylase domain carboxylates a biotin prosthetic group linked to a specific lysine residue in the biotin-carboxyl-carrier protein (BCCP) domain. Acetyl-coenzyme A activates reaction (1) by increasing the rate of bicarbonate-dependent ATP cleavage. In reaction (2), the BCCP domain donates the CO2 to pyruvate in a reaction catalyzed by the transcarboxylase domain (Attwood, P. V., Int. J. Biochem. Cell. Biol. 27:231-249 (1995)).
Pruvate carboxylase genes have been cloned and sequenced from: Rhizobium etli (Dunn, M. F., et al., J. Bacteriol. 178:5960-5970 (1996)), Bacillus stearothermophilus (Kondo, H., et al., Gene 191:47-50 (1997), Bacillus subtillis (Genbank accession no. Z97025), Mycobacterium tuberculosis (Genbank accession no. Z83018), and Methanobacterium thermoautotrophicum (Mukhopadhyay, B., J. Biol. Chem. 273:5155-5166 (1998). Pyruvate carboxylase activity has been measured previously in Brevibacterium lactofermentum (Tosaka, O., et al., Agric. Biol. Chem. 43:1513-1519 (1979)) and Corynebacterium glutamicum (Peters-Wendisch, P. G., et al., Microbiology 143:1095-1103 (1997)).
Previous research has indicated that the yield and productivity of the aspartate family of amino acids depends critically on the carbon flux through anaplerotic pathways (Vallino, J. J., and Stephanopoulos, G., Biotechnol. Bioeng. 41:633-646 (1993)). On the basis of the metabolite balances, it can be shown that the rate of lysine production is less than or equal to the rate of oxaloacetate synthesis via the anaplerotic pathways.
The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding a pyruvate carboxylase polypeptide having the amino acid sequence in FIG. 1 (SEQ ID NO:2) or the amino acid sequence encoded by the clone deposited in a bacterial host as ATCC Deposit Number PTA982. The nucleotide sequence determined by sequencing the deposited pyruvate carboxylase clone, which is shown in FIG. 1 (SEQ ID NO:1), contains an open reading frame encoding a polypeptide of 1140 amino acid residues which has a deduced molecular weight of about 123.6 kDa. The 1140 amino acid sequence of the predicted pyruvate carboxylase protein is shown in FIG. 1 and in SEQ ID NO:2.
Thus, one aspect of the invention provides an isolated nucleic acid molecule comprising a polynucleotide having a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence encoding the pyruvate carboxylase polypeptide having the complete amino acid sequence in SEQ ID NO:2; (b) a nucleotide sequence encoding the pyruvate carboxylase polypeptide having the complete amino acid sequence encoded by the clone contained in ATCC Deposit No. PTA982; and (c) a nucleotide sequence complementary to any of the nucleotide sequences in (a) or (b) above.
Further embodiments of the invention include isolated nucleic acid molecules that comprise apolynucleotide having anucleotide sequence at least 90% identical, and more preferably at least 95%, 97%, 98% or 99% identical, to any of the nucleotide sequences in (a), (b) or (c) above, or a polynucleotide which hybridizes under stringent hybridization conditions to a polynucleotide having a nucleotide sequence identical to a nucleotide sequence in (a), (b) or (c), above. The polynucleotide which hybridizes does not hybridize under stringent hybridization conditions to a polynucleotide having a nucleotide sequence consisting of only A residues or of only T residues.
The present invention also relates to recombinant vectors which include the isolated nucleic acid molecules of the present invention and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells and for using them for production of pyruvate carboxylase polypeptides or peptides by recombinant techniques.
The invention further provides an isolated pyruvate carboxylase polypeptide having amino acid sequence selected from the group consisting of: (a) the amino acid sequence of the pyruvate carboxylase polypeptide having the amino acid sequence shown in FIG. 1 (SEQ ID NO:2); and (b) the amino acid sequence of the pyruvate carboxylase polypeptide having the complete amino acid sequence encoded by the clone contained in ATCC Deposit No. PTA982. The polypeptides of the present invention also include polypeptides having an amino acid sequence with at least 90% similarity, more preferably at least 95% similarity to those described in (a) or (b) above, as well as polypeptides having an amino acid sequence at least 70% identical, more preferably at least 90% identical, and still more preferably 95%, 97%, 98% or 99% identical to those above.